FACS assays were performed as previously described [3 (link)]. TMRE (10 nM) was used to measure mitochondrial membrane potential; C11-BODIPY (50 μM) was applied to detect lipid ROS; PGSK (20 μM) was used to detect intracellular iron; Calcein-AM (50 nM) was used to determine cell viability; Cytosolic LIP was measured according to the methods described previously [56 (link)]. Briefly, cells were trypsinized, washed twice with 0.5 ml of PBS (Beyotime, C0221A), and then stained with calcein-AM (50 nM) for 15 min at 37°C. After washing twice with PBS, one-half of the stained HUVECs were incubated with DFP (100 μM) for 1 h at 37°C. Calcein was excited at 488 nm, and fluorescence was measured at 525 nm. The difference in the mean fluorescence with and without DFP incubation reflects the amount of cytosolic LIP. All FACS experiments were performed on BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) and results were analyzed using the BD FACS Software (San Jose, CA, USA).